Atmospheric Pressure Photoionization Fourier Transform Ion

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Anal. Chem. 2006, 78, 5906-5912

Atmospheric Pressure Photoionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Complex Mixture Analysis Jeremiah M. Purcell,† Christopher L. Hendrickson,† Ryan P. Rodgers,† and Alan G. Marshall*,†

National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005

We have coupled atmospheric pressure photoionization (APPI) to a home-built 9.4-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Analysis of naphtho[2,3-a]pyrene and crude oil mass spectra reveals that protonated molecules, deprotonated molecules, and radical molecular ions are formed simultaneously in the ion source, thereby complicating the spectra (>12 000 peaks per mass spectrum and up to 63 peaks of the same nominal mass), and eliminating the “nitrogen rule” for nominal mass determination of number of nitrogens. Nevertheless, the ultrahigh mass resolving power and mass accuracy of FT-ICR MS enable definitive elemental composition assignments, even for doublets as closely spaced as 1.1 mDa (SH313C vs 12C4). APPI efficiently ionizes nonpolar compounds that are unobservable by electrospray and allows nonpolar sulfur speciation of petrochemical mixtures. Advancements in atmospheric pressure ionization (API) techniques have broadened the analytical possibilities for mass spectrometry. Notably, electrospray ionization (ESI)1 and atmospheric chemical ionization (APCI)2 have expanded the application of mass spectrometry to the biological and pharmaceutical sciences.3,4 Both ESI and APCI mechanisms attach a charge to the analyte and ionization efficiency correlates with analyte polarity. Electrospray ionization of a neutral analyte typically occurs by addition or loss of a proton. The APCI charge carrier is the product of a corona discharge, typically CH5+ from methane, but can vary with different gas systems. These API techniques have the advantages of ready coupling with liquid chromatography (LC), can efficiently ionize polar species and to some extent less polar species, and are robust. However, nonpolar compounds are inaccessible by ESI and can be problematic for APCI. Atmospheric pressure photoionization (APPI) was initially introduced as a soft ionization method through direct photoionization5-8 and later with dopant-assisted ionization coupled to LC9 * To whom correspondence should be addressed. Telephone: 1-850-644-0529. Fax: 1-850-644-1366. E-mail: [email protected]. † Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306-4390. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (2) Carroll, D. I.; Dzidid, I.; Stillwell, R. N.; Haegele, K. D.; Horning, E. C. Anal. Chem. 1975, 47, 2369-2373. (3) Smith, R. D. Int. J. Mass Spectrom. 2000, 200, 509-544. (4) Rosenberg, E. J. Chromatogr., A 2003, 1000, 841-889. (5) Revel’skii, I. A.; Yashin, Y. S.; Voznesenskii, V. N.; Kurochkin, V. K.; Kostyanovskii, R. G. IIzv. Akad. Nauk SSSR, Ser. Khim. 1986, 9, 19871992.

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and can produce ions of low polarity and even nonpolar species not efficiently ionized by ESI and APCI. Field desorption ionization10,11 can also produce ions from nonpolar species, but (less conveniently) at less than atmospheric pressure. An APPI ion source typically uses a vacuum ultraviolet (VUV) gas discharge lamp (e.g., krypton at ∼120 nm) and can produce radical molecular ions from species with first ionization energies (IE) below the photon energy. However, some typical LC solvents (acetonitrile, methanol, water) deplete much of the photon flux resulting in poor analyte ionization efficiency.12-15 Poor ionization efficiency by direct photoionization is problematic. Robb et al. have shown that the addition of a dopant, toluene, increases sensitivity by promoting proton-transfer reactions and charge exchange reactions.9 Robb eluted four model compounds with and without a dopant and noted a 100-fold increase in signal with a dopant for some compounds. Consequently, most APPI configurations have coupled LC to APPI with a toluene dopant.16 The dopant is introduced directly into the solvent flow postcolumn or infused into a stream of hot gas through the auxiliary gas port of the APPI source heated nebulizer. Toluene has a first IE of 8.3 eV17 (lower than that of the photons from the VUV lamp) and is typically infused at a flow rate that results in a relative molar concentration much higher than that of the analyte. The combination of low first ionization energy and high molar concentration increases the statistical probability that an analyte ion will form because the abundant dopant radical molecular ions collide reactively with the analyte.9,18 (6) Revel’skii, I. A.; Yashin, Y. S.; Kurochkin, V. K.; Kostyanovskii, R. G. Zavodskaya Laboratoriya 1991, 57, 1-4. (7) Syage, J. A.; Evans, M. D.; Hanold, K. A. Am. Lab. 2000, 32, 24-29. (8) Syage, J. A.; Evans, M. D. Spectroscopy 2001, 16, 14-21. (9) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 3653-3659. (10) Schaub, T. M.; Hendrickson, C. L.; Qian, K.; Quinn, J. P.; Marshall, A. G. Anal. Chem. 2003, 75, 2172-2176. (11) Schaub, T. M.; Hendrickson, C. L.; Quinn, J. P.; Rodgers, R. P.; Marshall, A. G. Anal. Chem. 2005, 77, 1317-1324. (12) Raffaelli, A.; Saba, A. Mass Spectrom. Rev. 2003, 22, 318-331. (13) Kauppila, T. J.; Bruins, A. P.; Kostiainen, R. J. Am. Soc. Mass Spectrom. 2005, 16, 1399-1407. (14) Robb, D. B.; Blades, M. W. J. Am. Soc. Mass Spectrom. 2005, 16, 12751290. (15) Robb, D. B.; Blades, M. W. J. Am. Soc. Mass Spectrom. 2006, 17, 130-138. (16) Tubaro, M.; Marotta, E.; Seraglia, R.; Traldi, P. Rapid Commun. Mass Spectrom. 2003, 17, 2423-2429. (17) Lias, S. G. NIST, 2003. (18) Kauppila, T. J.; Kuuranne, T.; Meurer, E. C.; Eberlin, M. N.; Kotiaho, T.; Kostiainen, R. Anal. Chem. 2002, 74, 5470-5479. 10.1021/ac060754h CCC: $33.50

© 2006 American Chemical Society Published on Web 07/04/2006

There are two primary ionization products for a neutral analyte in the APPI source. With toluene dopant, if the proton affinity of the analyte is higher than the proton affinity of the benzyl radical, a protonated molecule can form.9,18 If the electron affinity of the toluene radical cation is higher than the electron affinity of the analyte (lower or equal ionization energy than toluene), a radical molecular ion can form. The possibility of forming two ion types from a single analyte can further complicate an already complex spectrum. High mass resolving power and mass accuracy are particularly essential for APPI MS. Greig et al. first coupled APPI to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry and applied it to the analysis of corticosteroids.19 In this work, we couple APPI with a 9.4-T FT-ICR mass spectrometer20 to evaluate model compounds as well as complex mixtures. We chose petroleum crude oil for demonstration, because it contains both polar and nonpolar constituents and represents the most complex natural mixture over a relative abundance dynamic range of ∼104. We further demonstrate that the ultrahigh mass resolving power and ultrahigh mass accuracy of FT-ICR MS21 are essential for analysis of complex mixtures by APPI mass spectrometry. EXPERIMENTAL METHODS Solvents and Compounds. All solvents were HPLC grade and purchased from Fisher. Naphtho[2,3-a]pyrene was purchased from Sigma-Aldrich and dissolved in toluene or an isomeric mixture of hexanes to produce a 3 mM stock solution. Two serial dilutions resulted in a 30 µM final concentration in toluene or hexane. Crude Oil. Each crude oil was supplied by ExxonMobil, and a sample (2.5 g) was fractionated according to the saturatesaromatics-resins-asphaltenes method.22 The crude oil was dissolved in 20 mL of toluene and rotary vacuum-evaporated to ∼5 mL volume to remove the volatiles. The sample was then completely dried under a stream of nitrogen gas. The dried sample was dissolved in n-hexane (25 mL) and gravity filtered through Whatman 2V grade paper to remove the asphaltenes. The filtrate (maltenes) was rotary vacuum-evaporated to 5 mL volume, absorbed onto aluminum oxide (Al2O3, 5.2 g), and dried under a stream of nitrogen gas with gentle stirring. The alumina was then packed on top of neutral alumina (15.0 g) in an 11 × 300 mm open column. The aliphatics were eluted with hexane (80 mL) and the aromatics subsequently eluted with toluene (80 mL) and the resins with 80:20 toluene/methanol (80 mL). The aromatic sample was rotary vacuum-evaporated to dryness and weighed (1.13 g) and redissolved in toluene (11 mL) to produce a stock solution of 100 mg/mL. The stock solution was further diluted to 1 mg/mL in toluene for analysis. APPI Source. The APPI source was supplied by Thermo Electron. The vaporized analyte gas stream flows orthogonally to the mass spectrometer inlet (heated metal capillary) and the krypton vacuum UV lamp (Figure 1). The source was mounted (19) Greig, M. J.; Bolanos, B.; Quenzer, T.; Bylund, J. M. R. Rapid Commun. Mass Spectrom. 2003, 17, 2763-2768. (20) Senko, M. W.; Hendrickson, C. L.; PasaTolic, L.; Marto, J. A.; White, F. M.; Guan, S. H.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (21) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (22) Vazquez, D.; Mansoori, G. A. J. Pet. Sci. Eng. 2000, 26, 49-55.

Figure 1. Two-dimensional layout of the APPI ion source. For simplicity, the vacuum UV lamp is drawn along the z axis with the heated metal capillary. In practice, the lamp is along the x axis so that the three assemblies are mutually orthogonal.

to a home-built adapter to interface to the first differentially pumped stage of the 9.4-T FT-ICR mass spectrometer through a heated metal capillary. A Harvard stainless steel syringe (8 mL) and syringe pump were utilized to deliver solution to the heated nebulizer of the APPI source. In the APPI source, solvent flow rate was 50-100 µL/min; the nebulizer heater was operated at 250-350 °C with carbon dioxide as the sheath gas at 80 psi, and the auxiliary gas port was plugged. 9.4-T FT-ICR MS. All experiments were performed with a home-built FT-ICR mass spectrometer equipped with a passively shielded Oxford 9.4-T superconducting magnet.20,23 The mass spectrometer was controlled by a modular ICR data system.24,25 Ions were produced by the external APPI source and traversed the heated metal capillary to the first stage of vacuum pumping into a skimmer region. The skimmer provides a conductance limit to the second stage of differential pressure where the ions enter the first radio frequency (rf)-only octopole. In the first octopole, ions were accumulated (5-10 s)26 before transfer through a quadrupole (not operated in mass-resolving mode) into a second rf-only octopole where they were collisionally cooled (10-20 ms) with helium before transfer through an rf-only octopole to a 10cm-diameter, 30-cm-long open cylindrical Penning ion trap. The octopole ion guides (1.6-mm titanium rods with a 4.8-mm i.d.) were typically operated between 1.5 and 2.0 MHz and 190 < Vp-p < 240 V rf amplitude. Broadband frequency-sweep excitation (∼90600 kHz at a sweep rate of 150 Hz/µs and a 190 V peak-to-peak amplitude) accelerated the ions to a detectable cyclotron orbital radius. Ion cyclotron resonant frequencies were detected from induced current on two opposed detection electrodes of the ICR trap. Multiple (9-400) time-domain acquisitions were summed for each sample, Hanning-apodized, and zero-filled once before (23) Hakansson, K.; Chalmers, M. J.; Quinn, J. P.; McFarland, M. A.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2003, 75, 3256-3262. (24) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1839-1844. (25) Blakney, G. T.; Chalmers, M. J.; Lam, T. T.; Emmett, M. R.; Hendrickson, C. L.; Marshall, A. G. Abstract of Papers, 51st Conference on Mass Spectrometry and Allied Topics, American Society for Mass Spectrometry, Montre´al, PQ, Canada, 2003. (26) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976.

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Kendrick Mass Analysis. Because crude oil consists primarily of homologous series differing by nCH2 (n is a positive integer), it is convenient to convert the IUPAC mass to Kendrick mass.29,30

Kendrick mass ) IUPAC mass × (14/14.01565) (1) The Kendrick masses for members of a homologous alkylation series differ by increments of exactly 14 Da and have the same Kendrick mass defect (KMD).

KMD ) (Kendrick nominal mass Figure 2. Class distribution from an APPI positive-ion FT-ICR mass spectrum of Middle East crude oil.

Kendrick exact mass) × 1000 (2)

fast Fourier transform and magnitude calculation.27 Negative ion data were collected with similar parameters and appropriate polarity changes. All observed ions were singly charged, based on the unit m/z separation between 12Cn and 13C112Cn-1 isotopic variants of the same elemental composition.28 Therefore, mass spectral peak positions are reported in daltons rather than as m/z.

The data may then be sorted by KMD in a spreadsheet to facilitate assignment of elemental composition of each homologous series. RESULTS AND DISCUSSION FT-ICR MS can simultaneously analyze ions spanning several decades in mass-to-charge ratio (m/z), over a vertical dynamic

Figure 3. APPI positive-ion FT-ICR mass spectra of 30 µM naphtho[2,3-a]pyrene in toluene (top) and hexanes (bottom). Top: The insets show the two kinds of ions formed in the APPI source region: protonated molecules and radical molecular cations. Nine acquisitions were summed with an external ion accumulation of 5 s each, resulting in a SNR of 2300. Bottom: The insets show the reduction in formation of the protonated molecule in the absence of a dopant. Also, the SNR of 200 is significantly less than in Figure 2. Nine acquisitions were summed with an external ion accumulation of 10 s each. 5908

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Figure 4. APPI FT-ICR mass scale-expanded segment for a South American crude oil. The mass doublets document the requirement for ultrahigh mass resolving power with an APPI source for complex mixture analysis. The 3.4-mDa mass doublet corresponds to species differing by C3 vs SH4 and the 4.5-mDa mass doublet to 12CH vs 13C. Two hundred acquisitions were summed with an ion accumulation of 3 s each. Starred peaks were assigned to elemental compositions not shown in the figure.

Figure 5. APPI FT-ICR mass scale-expanded segment of a highsulfur Middle East crude oil, showing a very close 1.1-mDa mass doublet, 12C4 vs SH313C. Two hundred acquisitions were summed with an external ion accumulation of 5 s each. Starred peaks were assigned to elemental compositions not shown in the figure.

range of up to 104, with ultrahigh resolving power and mass accuracy.21 Time-of-flight and quadrupole mass spectrometers have lower mass resolving power and usually require an LC preseparation step prior to mass analysis of complex mixtures. Therefore, FT-ICR MS can negate the need for a preseparation step by combining both resolution and mass measurement in one step. Although crude oil is the most compositionally complex organic mixture (over a dynamic range of 104); ESI FT-ICR MS has enabled the detailed speciation of its polar components.31,32 However, because the electrospray ionization mechanism involves proton-transfer reactions, it selectively ionizes acids (to produce negative ions) or bases (to produce positive ions). Thus, a limitation of ESI is that it will most efficiently ionize the most acidic/basic species. Atmospheric pressure photoionization, on the other hand, can ionize both polar and nonpolar compounds. Because petroleum crude oil is composed ∼90% of hydrocarbons,

APPI generates mass spectral signals for species not accessible by ESI or APCI. For example, Figure 2 shows heteroatom class relative abundances for ions in a positive-ion APPI FT-ICR mass spectrum of a Middle East crude oil. The starred classes are nonpolar classes observed by APPI and are not detected by ESI, the most notable of which are those that contain sulfur. Sulfur speciation is particularly important to the petroleum refining industry due to continued regulatory decreases in the allowable sulfur levels for petroleum products. A detailed discussion of the species observed in ESI and APPI, their respective class-based ionization trends, and APPI ionization mechanisms in complex petroleum matrixes will be reported elsewhere. Model Compounds. Figure 3 shows APPI FT-ICR positiveion mass spectra of a polycyclic aromatic hydrocarbon, naphtho[2,3-a]pyrene, dissolved in toluene (top) and a mixture of isomeric hexanes (bottom) and injected directly into the APPI source. Both spectra were collected under the same instrumental conditions except for a doubled ion accumulation period for Figure 3 (bottom) to enhance the signal-to-noise ratio. Even so, the signal-to-noise ratio in substantially lower for the hexanes sample. The higher signal-to-noise ratio in Figure 3 (top) is attributed to charge exchange between the toluene dopant and the analyte. In this example, ionization efficiency is enhanced 20-fold by addition of the dopant. The mass scale-expanded insets in Figure 3 reveal another difference between the spectra. At nominal mass 303 Da, the protonated molecule ([C24H14 + H]+) relative abundance in hexanes (Figure 3b) is significantly lower than in toluene (Figure 3, top). In Figure 3 (top), the signal from the 12C24 protonated molecule at nominal mass 303 is comparable in magnitude to that for the 13C112C23 radical cation. Lower resolving power mass analyzers would not resolve that doublet, and the resulting broadened and asymmetrical peak would yield poor mass accuracy leading to ambiguity in peak assignment. Also in Figure 3 (top), three species are detected at nominal mass 304 Da. The use of a dopant (toluene) clearly increases signal-to-noise ratio, but at the cost of increased spectral complexity due to formation of two ion types (radical molecular ions and protonated molecules), thereby increasing the need for ultrahigh resolving power for reliable assignment of elemental compositions. Nitrogen Rule. At nominal mass accuracy, the “nitrogen rule”33 states that an odd-electron ion (e.g., M+•) has an even (odd) nominal mass if it contains an even (odd) number of nitrogen atoms.” Conversely, an even-electron ion (e.g., (M + H)+ or (M - H)-) has an odd (even) nominal mass if it contains an even (odd) number of nitrogen atoms. Thus, it is possible to determine whether the number of nitrogens is even or odd based on appearance of a mass spectral signal at even or odd nominal mass, provided that all ions are either even-electron (as in electrospray ionization or matrix-assisted laser desorption/ionization) or odd(27) Marshall, A. G.; Verdun, F. R. Fourier Transforms in NMR, Optical, and Mass Spectrometry: A User’s Handbook; Elsevier: Amsterdam, 1990. (28) Senko, M. W.; Beu, S. C.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1995, 6, 52-56. (29) Kendrick, E. Anal. Chem. 1963, 35, 2146-2154. (30) Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G.; Qian, K. Anal. Chem. 2001, 73, 4676-4681. (31) Marshall, A. G.; Rodgers, R. P. Acc. Chem. Res. 2004, 37, 53-59. (32) Rodgers, R.; Schaub, T.; Marshall, A. Anal. Chem. 2005, 77, 20A-27A. (33) McLafferty, F. W.; Turececk, F. Interpretation of Mass Spectra, 4th ed.; University Science Books: Mill Valley, CA, 1993.

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Figure 6. Negative ion APPI FT-ICR broadband mass spectrum of a South American crude oil. Bottom: Across a 400-Da mass window, 12 449 unique elemental compositions (a new record for a single mass spectrum) were assigned (> 99% deprotonated molecules), based on an average mass resolving power of ∼400 000 and an rms mass accuracy of 260 ppb. Top: At a S/N ratio >8σ of baseline noise, there are 63 spectral peaks of nominal mass 377 Da of which unique elemental compositions could be assigned to 62 (see Table 1).

electron (as in electron ionization). However, because APPI can produce both even- and odd-electron ions in the same spectrum, the nitrogen rule can no longer be used to determine the number of nitrogens based on nominal mass alone. Again, ultrahigh resolution and mass accuracy are needed to derive the correct elemental composition. Complex Mixture Analysis. Formation of both protonated molecules and radical ions obviously increases the number of peaks per nominal mass. In Figure 3 (top), at nominal mass 303, the difference between [C23H1413C]+• and [C24H14+ H]+ is 4.5 mDa (13C vs CH), requiring mass resolving power of at least 130 000 (and proportionately higher at higher mass or for unequal relative abundance) for correct elemental composition assignment. In a complex mixture, FT-ICR MS routinely achieves ultrahigh mass resolving power (e.g., 400 000 resolving power at 400 Da), m/∆m50%, in which ∆m50% is the mass spectral peak full width at half-maximum peak height.21 For example, Figure 4 shows a mass scale-expanded segment of a positive-ion APPI FT-ICR mass spectrum of a South American crude oil. A single crude oil mass spectrum can contain thousands of peaks.34,35 The 3.4-mDa 5910

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separation seen in Figure 4 is the mass difference between C3 and SH4, a common mass doublet in crude oil. That mass difference is also seen by electrospray ionization because both species are amenable to proton-transfer reactions and can produce protonated molecules. Furthermore, the formation of both radical cations and protonated molecules in the APPI source can produce an additional 4.5-mDa split (13C vs CH). Figure 5 shows a mass scale-expanded segment of a positiveion APPI FT-ICR mass spectrum of a Middle East crude oil known to be high in sulfur content.36 The combination of high sulfur content and two ionization pathways produces yet another mass doublet, separated by only 1.1 mDa, corresponding to the mass difference between C4 and SH313C from the protonated molecule [C24H29N1S113C1 + H]+ and the radical molecular ion [C28H27N1]+•. (34) Qian, K.; Robbins, W. K.; Hughey, C. A.; Cooper, H. J.; Rodgers, R. P.; Marshall, A. G. Energy Fuels 2001, 15, 1505-1511. (35) Hughey, C. A.; Rodgers, R. P.; Marshall, A. G. Anal. Chem. 2002, 74, 41454149. (36) Altgelt, K. H.; Boduszynski, M. M. Composition and analysis of heavy petroleum fractions; Marcel Dekker: New York, 1994.

Table 1. Elemental Compositions Assigned to Peaks in the Negative-Ion APPI FT-ICR Mass Spectral Segment Shown in Figure 6a peak no.

elemental composition

measured mass

calculated mass

ppm error

peak no.

elemental composition

measured mass

calculated mass

ppm error

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

C24H15O4S2 C20H15O9S1 C24H15O6S1 C21H19O6S2 C28H15O3S1 C24H15O8 C21H19O8S1 C28H15O5 C25H19O5S1 C22H23O5S2 C29H19O2S1 C25H19O7 C22H23O7S1 C19H27O7S2 C29H19O4 C26H23O4S1 C23H27O4S2 C26H23O6 C25H24N1O3S113C1 C23H27O6S1 C20H31O6S2 C30H23O3 C27H27O3S1 C24H31O3S2 C20H31O8S1 C29H24N1O213C1 C27H27O5 C26H28N1O2S113C1 C24H31O5S1 C21H35O5S2 C31H27O2

431.041 59 431.044 20 431.059 44 431.062 72 431.074 77 431.077 23 431.080 55 431.092 42 431.095 80 431.099 21 431.111 12 431.113 61 431.116 98 431.120 43 431.128 86 431.132 25 431.135 59 431.150 00 431.151 35 431.153 36 431.156 75 431.165 28 431.168 63 431.172 03 431.174 49 431.184 46 431.186 38 431.187 69 431.189 76 431.193 11 431.201 61

431.041 72 431.044 23 431.059 48 431.062 85 431.074 74 431.077 24 431.080 61 431.092 50 431.095 87 431.099 24 431.111 12 431.113 63 431.117 00 431.120 37 431.128 88 431.132 25 431.135 62 431.150 01 431.151 59 431.153 38 431.156 75 431.165 27 431.168 64 431.172 01 431.174 51 431.184 61 431.186 40 431.187 98 431.189 77 431.193 14 431.201 65

-0.31 -0.06 -0.10 -0.31 0.07 -0.03 -0.14 -0.18 -0.16 -0.07 -0.01 -0.04 -0.04 0.14 -0.05 -0.01 -0.08 -0.03 -0.56 -0.05 -0.01 0.03 -0.02 0.05 -0.05 -0.34 -0.04 -0.67 -0.02 -0.07 -0.10

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

C28H31O2S1 C24H31O7 C23H32N1O4S113C1 C21H35O7S1 C30H28N1O113C1 C28H31O4 C27H32N1O1S113C1 C25H35O4S1 C22H39O4S2 C29H35O1S1 C27H32N1O313C1 C25H35O6 C22H39O6S1 C31H31N2 C31H32N113C1 C29H35O3 C28H36N1S113C1 C26H39O3S1 C28H36N1O213C1 C26H39O5 C23H43O5S1 C30H39O2 C27H43O2S1 C29H40N1O113C1 C27H43O4 C24H47O4S1 C31H43O1 C28H47O1S1 C30H44N113C1 C28H47O3 C29H51O2

431.204 99 431.207 46 431.209 18 431.210 84 431.221 03 431.222 76 431.224 11 431.226 12 431.229 51 431.241 29 431.242 12 431.243 86 431.247 26 431.249 10 431.257 37 431.259 11 431.260 53 431.262 46 431.278 43 431.280 20 431.283 61 431.295 46 431.298 79 431.314 85 431.316 59 431.319 97 431.331 84 431.335 20 431.351 22 431.353 01 431.389 48

431.205 02 431.207 53 431.209 11 431.210 90 431.220 99 431.222 78 431.224 36 431.226 15 431.229 52 431.241 41 431.242 12 431.243 91 431.247 28 431.249 27 431.257 38 431.259 17 431.260 75 431.262 54 431.278 51 431.280 30 431.283 67 431.295 55 431.298 92 431.314 89 431.316 68 431.320 05 431.331 94 431.335 31 431.351 28 431.353 07 431.389 45

-0.08 -0.16 0.17 -0.13 0.09 -0.05 -0.59 -0.08 -0.03 -0.28 -0.01 -0.12 -0.05 -0.40 -0.02 -0.14 -0.51 -0.18 -0.18 -0.23 -0.14 -0.22 -0.31 -0.10 -0.22 -0.20 -0.23 -0.26 -0.14 -0.14 0.06

a All elemental compositions are for the deprotonated molecule, (M - H)-. Note that measured and calculated masses are uniformly identical to six places and differ only at the sub-ppm level (last two digits of each entry).

That doublet is not seen in ESI spectra because radical cations are not observed by ESI for crude oil samples. Negative Ions. Negative ions are formed along with positive ions in the APPI source and may be detected with appropriate instrument polarity changes. Figure 6 (bottom) is a negative-ion APPI FT-ICR broadband mass spectrum of a South American crude oil and further demonstrates the remarkable and necessary analytical power of FT-ICR MS. Across the spectrum, unique elemental compositions could be assigned to 12 449 spectral peaks: the most compositionally complex resolved mass spectrum to date. Within the 400-Da mass window, an average mass resolving power of ∼400 000 and an rms mass accuracy of 260 parts-per-billion (ppb) were achieved. An example of the complexity is shown in the mass scale expansion (top). There are 63 spectral peaks with magnitude exceeding 8σ of rms baseline noise, and unique elemental compositions could be assigned to 62 of them (see Table 1) based solely on mass accuracy and Kendrick analysis. Mass Accuracy. Figures 7 and 8 graphically demonstrate the unrivaled mass accuracy of FT-ICR MS, by showing the relation between peak magnitude and mass error (i.e., difference between experimentally measured mass and the exact mass corresponding to the elemental composition assigned to that mass spectral peak). The precision in measurement of peak position should be linearly proportional to the mass spectral peak signal-to-noise ratio (SNR)

Figure 7. APPI FT-ICR mass spectral peak magnitude vs mass error (measured mass minus the exact mass for the assigned chemical formula) for the elemental compositions assigned to 12 449 spectral peaks from Figure 6. Ninety percent of the peaks exhibit less than 500 ppb mass error. As predicted,37 mass accuracy increases with increasing mass spectral S/N ratio.

and the square root of the number of data points per peak width.37 Figure 7 shows that the mass error does increase as peak signalto-noise ratio decreases as expected; nevertheless, 90% of the peaks exhibit less than 500 ppb mass error. Figure 8 shows a more conventional mass error distribution, based on counting the number of peaks in each 50 ppb mass error “bin”. The errors are (37) Chen, L.; Cottrell, C. E.; Marshall, A. G. Chemom. Intell. Lab. Syst. 1986, 1, 51-58.

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CONCLUSIONS Atmospheric pressure photoionization is useful for analysis of low-polarity and nonpolar compounds. A dopant is typically necessary to increase sensitivity by promotion of proton-transfer and charge exchange reactions. Toluene works well because it can act as a proton donor (or, the toluene radical cation, as an electron acceptor) and participate in reactions that produce both cations and anions. Protonated molecules (and deprotonated molecules) and radical molecular ions are formed simultaneously. As a result, APPI can add complexity to mass spectra. FT-ICR MS overcomes that complication. APPI FT-ICR MS is uniquely suited to analysis of complex petrochemical mixtures that naturally contain a large proportion of low-polarity or nonpolar aromatic hydrocarbons and for analysis of fullerene mixtures.38 Figure 8. Mass error distribution for the 12 449 spectral peaks from Figure 6. Each bar represents the number of assigned masses within a 50 ppb “bin” mass error range. At half-maximum height, the errors span a range of (200 ppb.

Gaussian-distributed, with an rms deviation of (200 ppb. The data in Figures 7 and 8 constitute the most definitive measures of broadband mass measurement accuracy, especially at low signalto-noise ratio. (38) Talyzin, A. V.; Tsybin, Y. O.; Purcell, J. M.; Schaub, T. M.; Shulga, Y. M.; Noreus, D.; Sato, T.; Dzwilewski, A.; Sundqvist, B.; Marshall, A. G. J. Phys. Chem. A 2006.

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Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

ACKNOWLEDGMENT The authors thank Michael Senko, Rohan Thakur, and Julie Horner at Thermo Electron for lending the APPI source. Also, we thank John P. Quinn and Dan McIntosh for the design and fabrication of an APPI source adapter. This work was supported by NSF DMR-00-84173, Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, FL 323104005.

Received for review April 20, 2006. Accepted May 23, 2006. AC060754H